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An fMRI Study Examining Effects of Acute D-Cycloserine During Symptom Provocation in Spider Phobia


Robin L. Aupperle, PhD, Lisa R. Hale, PhD, Rebecca J. Chambers, MA, Sharon E. Cain, MD, Frank X. Barth, DO, Susan C. Sharp, DO, Douglas R. Denney, PhD, and Cary R. Savage, PhD


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CNS Spectr. 2009;14(10):556-571

Faculty Affiliations and Disclosures

Dr. Aupperle is postdoctoral research fellow in the Department of Psychiatry at the University of California, San Diego. Dr. Hale is research assistant professor in the Department of Neurology at University of Kansas Medical Center in Kansas City, KS. Ms. Chambers is research assistant at Hoglund Brain Imaging Center in Kansas City, KS. Dr. Cain is clinical professor in the Department of Psychiatry at University of Kansas Medical Center in Kansas City, KS. Dr. Barth is clinical assistant professor in the Department of Psychiatry at University of Kansas Medical Center in Kansas City, KS. Dr. Sharp is clinical assistant professor in the Department of Psychiatry at University of Kansas Medical Center in Kansas City, KS. Dr. Denney is professor in the Department of Psychology at University of Kansas in Lawrence, KS. Dr. Savage is professor in the Department of Psychiatry at University of Kansas Medical Center in Kansas City, KS.

Faculty Disclosures: Dr. Barth receives grant/research support from Abbott. Dr. Denney is a consultant to Teva Neuroscience. Dr. Savage is a consultant to Merck. Drs. Aupperle, Hale, Chambers, Cain, and Sharp report no affiliations with or financial interest in any organiztion that may pose a conflict of interest.

Funding/Support: This research was funded by the University of Kansas Medical Center Clinical Pilot Research Grant, Hoglund Brain Imaging Center Pilot Research Fund, American Psychological Association Dissertation Scholarship, and American Psychological Foundation Research Scholarship. Hoglund Brain Imaging Center is supported by a gift from Forrest and Sally Hoglund.

Acknowledgments: The authors would like to thank Michael Davis, PhD for his advice during the planning stages of this project.

Abstract

Background: Exposure-based therapy for anxiety disorders is believed to operate on the basis of fear extinction. Studies have shown acute administration of D-cycloserine (DCS) enhances fear extinction in animals and facilitates exposure therapy in humans, but the neural mechanisms are not completely understood. To date, no study has examined neural effects of acute DCS in anxiety-disordered populations.
Methods: Two hours prior to functional magnetic resonance imaging scanning, 23 spider-phobic and 23 non-phobic participants were randomized to receive DCS 100 mg or placebo. During scanning, participants viewed spider, butterfly, and Gaussian-blurred baseline images in a block-design paradigm. Diagnostic and treatment groups were compared regarding differential activations to spider versus butterfly stimuli.
Results: In the phobic group, DCS enhanced prefrontal (PFC), dorsal anterior cingulate (ACC), and insula activations. For controls, DCS enhanced ventral ACC and caudate activations. There was a positive correlation between lateral PFC and amygdala activation for the placebo-phobic group. Reported distress during symptom provocation was correlated with amygdala activation in the placebo-phobic group and orbitofrontal cortex activation in the DCS-phobic group.  
Conclusions: Results suggest that during initial phobic symptom provocation DCS enhances activation in regions involved in cognitive control and interoceptive integration, including the PFC, ACC, and insular cortices for phobic participants.

INTRODUCTION

Anxiety disorders are among the most commonly diagnosed psychiatric disorders, with an estimated prevalence of 18% in the United States.1 Both pharmacotherapy, such as selective serotonin reuptake inhibitors, and cognitive behavior therapies (CBT), such as exposure and response prevention (ERP), are effective in treating anxiety disorders. Exposure-based therapies involve systematic and repeated exposure to anxiety-provoking stimuli, leading to habituation and extinction of the fear response. Response rates of CBT for anxiety range from 33% to 89%, depending on the disorder.2 Although response rates are higher for anxiety disorders than other psychiatric disorders,3 there is still considerable room for improvement. Combining standard medications and exposure therapy has not been shown to increase efficacy above what is found for either treatment alone.2,4 Recent clinical trials indicate, however, that the partial N-methyl-d-aspartic acid (NMDA) agonist, D-cycloserine (DCS), may enhance the effectiveness of exposure therapy.

Fear conditioning, involving concurrent presentation of a neutral stimulus with a naturally feared, or unconditioned, stimulus (UCS), is often used in animal models. Fear extinction can be instigated by exposing the animal to the conditioned stimulus (CS) alone until the fear response decreases. Animal models indicate that NMDA receptors are involved in learning and memory consolidation, including the process of fear extinction. Acute administration of DCS has been shown to enhance extinction of fear to CS,5,6 increase generalization of extinction,7 and decrease reinstatement of fear.8 From animal research, it is known that DCS binds to glycine recognition sites of NMDA receptors.9 Activation of NMDA receptors triggers a cascade of events leading to long term potentiation (LTP), which is thought to serve as the biological basis for learning.10,11 Animal studies show that direct infusion of DCS into the basolateral amygdala (BLA) or hippocampus enhances learning.5,12 DCS has also been shown to facilitate neural events associated with LTP within these regions13,14 and to affect D-serine concentration within the medial frontal cortex.15

The first human study to investigate the use of DCS for enhancing exposure therapy was conducted in 2004 with height phobia.16 DCS provided prior to exposure sessions was associated with greater decrease in fear of heights post-treatment. Similar studies have demonstrated that acute DCS administration enhances exposure therapy for other anxiety disorders, including social anxiety, panic, and obsessive-compulsive disorder.17-20 Though studies for the most part agree, one clinical study failed to find a beneficial effect of DCS on ERP response.21 It has been suggested that there are critical parameters under which DCS shows beneficial effects, including smaller rather than larger doses and administration close in time to the exposure session.22

Only two recent neuroimaging studies have examined neural mechanisms of acute DCS administration in humans, both involving healthy control participants. Britton and colleagues23 used functional magnetic resonance imaging (fMRI) to examine effects of DCS 500 mg on amygdala activation during an emotional faces paradigm. Those administered DCS failed to exhibit the increase in amygdala response observed for placebo-treated participants. Kalisch and colleagues24 examined effects of DCS 500 mg, administered directly following fear conditioning and extinction, on neural correlates of fear memory. When contrasting activations for CS+ (face stimuli that had previously been paired with shock) and CS- (face stimuli that had not been paired with shock), DCS led to greater activation in the hippocampus, medial prefrontal cortex (PFC), orbitofrontal cortex (OFC), inferior frontal gyrus, and insula. No differences in amygdala activation were noted. Results partially support findings from animal research, suggesting DCS modulates activity in regions associated with fear processing. However, the decreased amygdala activity reported by Britton and colleagues23 and the lack of modulation in amygdala activity reported by Kalisch and colleagues24 is somewhat unexpected, considering findings from animal research showing that although direct infusion of DCS into the amygdala produces modulation of fear extinction, it does not impair fear-potentiated startle, which is thought to relate to amygdala activity.5,15,25

The discrepancies between animal and human research may be partially due to differential effects of DCS for non-primate and human populations. It is also possible that DCS has varying influence depending on the paradigm (ie, fear memory versus facial processing) and population (ie, clinical versus control). Investigation of DCS in clinical populations is needed to further elucidate neural mechanisms for DCS-augmented exposure therapy.  
Our study is the first to investigate neural correlates of acute DCS in an anxiety-disordered population. We used fMRI to examine patients with specific phobia and healthy controls, with and without DCS. The primary objective was to investigate potential neural correlates of DCS during initial phobic symptom provocation. There are various stages of fear processing in which DCS may influence neural processes, including initial symptom provocation, habituation, consolidation, and recall. Many animal studies have shown DCS to be effective when administered post-extinction trials, suggesting DCS may target the consolidation phase.26 However, other animal studies and all published human studies, have found benefits for DCS when administered prior to exposure to feared stimuli. Therefore, the current study serves as a preliminary investigation regarding potential influences of DCS on neural processes during initial exposure. Results are important for understanding neural mechanisms through which DCS influences fear processing and exposure-based therapy and have implications regarding future research and clinical use of DCS.  

Methods

Participants

Fifty-four right-handed participants between 18 and 55 years of age (27 phobic, 27 non-phobic) participated in this study. Participants provided written consent and the study was approved by the human subjects committee of University of Kansas Medical Center (KUMC). Participants were provided $50 compensation for the session. Participants reporting medical conditions unsuitable for MRI scanning or a history of neurological or developmental disorder, head injury, or substance dependence were excluded. Participants with any Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition Axis I psychopathology, other than phobia, were excluded. None of the participants were taking psychotropics for at least 5 years prior to participation and none had a history of receiving psychotherapy.

Clinical Measures

Clinical information was collected by a trained clinician and supervised by a licensed psychologist. Assessment included the Anxiety Disorders Interview Schedule,27 Spider Questionnaire (SPQ),28 behavioral avoidance task (BAT), Beck Depression Inventory (BDI-II),29 and the Wechsler Adult Intelligence Scale (WAIS-III) Vocabulary and Matrix Reasoning subtests.30 The procedure used for the BAT was based upon that described by Merckelbach and colleagues.31 Participants were asked to get as close to a live tarantula (held in a plexiglass box) as they were comfortable with. Performance was measured by a 13-point scale (0=distance to spider >8 feet, 12=holding spider in hand).  

Medication

Optimal dosing for DCS has not been established in human studies. Clinical studies suggest DCS 50–125 mg are effective in enhancing exposure therapy.17-20 However, D’Souza and colleagues32 was able to detect only trace levels of DCS in cerebrospinal fluid (CSF) after oral administration of 50 mg. A single oral dose of DCS 250 mg reaches peak CSF concentration after 2 hours and has a plasma half-life of 9 hours.33 A dose of DCS 100 mg was chosen for the present study to remain consistent with clinical investigations and to optimize availability to the central nervous system. DCS and placebo capsules were prepared by KUMC investigational pharmacy and administered 2 hours prior to testing at the KUMC General Clinical Research Center under medical supervision by a board certified psychiatrist. Separate block randomizations (12 participants/block) were conducted for phobic and non-phobic groups. Each participant had a 50% chance of receiving DCS. Investigators, test administrators, and participants were blind to group assignment.

An adverse symptom checklist (ASC) was administered to assess potential side effects. The checklist was adapted from that used by Storch and colleagues21 and assessed neurological, gastrointestinal, cardiovascular, and musculoskeletal symptoms. Participants rated how “bothersome” they found each of 27 symptoms, using a scale of 0 (not at all) to 3 (severe). Participants also indicated whether they believed they were given DCS or placebo.

fMRI Paradigm

Scans were conducted using a 3 Tesla head-only Siemens Allegra scanner (Erlangen, Germany) with a quadrature head coil. Following automated scout image acquisition and shimming procedures, T1-weighted anatomic images were acquired with a three-dimensional (3-D) spoiled gradient recalled sequence (repetition time/echo time [TR/TE]=23/4 ms, flip angle=8°, field of view [FOV]=256 mm, matrix=256x192, slice thickness=1 mm). Two gradient echo blood oxygen level dependent (BOLD) scans were acquired in 43 contiguous coronal slices, perpendicular to the anterior-posterior commissure plane (TR/TE=3000/40 ms, flip angle=90°, FOV=192 mm, matrix=64x64, slice thickness=3 mm, 0.5 skip, in-plane resolution=3x3 mm, 130 data points) and were set for whole-brain coverage. Visual stimuli were projected through 3D limited-view goggles (Resonance Technology, Inc., Northridge, CA) connected to the stimulus presentation program (Presentation, Neurobehavioral Systems Inc., Albany, CA).

The symptom provocation paradigm was patterned after previous functional imaging studies with spider phobia.34 Three condition types were included: phobic (spider pictures), non-phobic (butterfly pictures), and low-level baseline (blurred images). Spider and butterfly pictures were obtained from validated databases, such as the International Affective Picture System,35 and from internet and other published sources.36 A pre-study validation study in spider-fearful participants was used to identify the 60 most arousing (assessed by the Self Assessment Manikin)35 of a collection of 80 spider pictures for use in the current study. Blurred images were created by applying a Gaussian kernel to animal pictures so the objects were unidentifiable. Each functional run alternated between blocks of 10 pictures from phobic, non-phobic, and baseline conditions, each presented for 3 seconds. The order of blocks was counterbalanced within- and across-participants to prevent order effects and no pictures were repeated.

fMRI Behavioral Measures

After each fMRI run, participants were asked to report subjective units of distress (SUDS; scale 0–8). After scanning, participants were given a memory task with 30 pictures (15 spider; 15 butterfly) from the fMRI paradigm interspersed with 20 novel images (10 spider; 10 butterfly). Participants were asked to indicate whether or not they had seen each image in the scanner. Recognition discriminability (D’) was calculated using the following formula:
100x[1-(#false positives + #false negatives)/total #pictures)].

fMRI Data Analysis

fMRI data were analyzed using BrainVoyager QX.37 Following 3-D motion correction, correction parameters were detrended, z-transformed, and entered as variables of no interest in the multi-study general linear model (GLM). Motion >3 mm along any axis resulted in discard of that run. Motion consistent with the paradigm, causing visible artifact on single-study GLM, also resulted in discard of that run. Sinc-interpolated slice scan time correction, high pass filter temporal smoothing, and 3D spatial smoothing (4-mm Gaussian filter) were also conducted. Functional images were realigned to anatomic images and normalized to the BrainVoyager template, which conforms to the space defined by Talairach and Tournoux’s stereotaxic atlas.38

Multiple regression analyses with GLM were used to examine differences in BOLD response between experimental conditions. Regressors of interest were modeled with a hemodynamic response filter and entered into the multiple regression using random-effects. Contrasts between conditions were assessed with t statistics for each voxel. Resulting statistics were displayed as statistical parameter maps and overlaid on three-dimensional renderings of averaged anatomic images from participants. Voxel values were considered significant if they met P<.001 and a cluster size of 3 contiguous voxels. While the whole brain was inspected for significant activations, a priori regions of interest (ROIs) were specified based on previous research and were the focus of inspection. These regions included, bilaterally: amygdala, insula, anterior cingulate cortex (ACC), OFC, dorsolateral PFC (dlPFC), and hippocampus. In the tables displaying results from fMRI analyses, a priori ROIs are shown in bold-faced type. To assess diagnostic effects and validate the paradigm for examining DCS effects, a diagnosis (placebo-phobic>placebo-control) x condition (spider>butterfly) mixed model interaction was utilized. To assess DCS effects, a treatment (DCS>placebo) x condition (spider>butterfly) mixed model interaction was utilized within each diagnostic group (phobic, non-phobic). Secondary condition main effects were inspected to investigate neural responses of the four groups separately.

Correlation analyses were conducted within each phobic group (placebo, DCS) to examine the relationship between a priori ROIs and clinical/behavioral data, and amygdala and prefrontal ROIs (because of theorized inhibitory relationships).39-42 For these analyses, we used maximum voxel percent signal change for the phobic condition. The specific regions were identified for each group separately based upon condition main effects (spider>butterfly). Correlations were conducted using bivariate Pearson correlations and were considered significant if they met P<.05.  

Clinical and Behavioral Data Analysis

Demographic, clinical, and fMRI behavioral data were analyzed using SPSS.43 For continuous variables, two-way analyses of variance (ANOVAs) were used to identify main effects and interactions for diagnosis and treatment. Fisher’s exact tests were used to identify differences between groups regarding categorical variables.

Results

Clinical and Demographic Data

Fifty-four participants enrolled in the study. Eight were excluded: one because of claustrophobia symptoms in the scanner; three because of scanner artifact; one because of motion >3mm; three because of paradigm-consistent motion (head movement only during spider conditions) which could not be corrected.44-46 Therefore, 46 participants were included in analyses (23 phobic [12 placebo, 11 DCS], 23 control [11 placebo, 12 DCS]).

Demographic and clinical data are included in Table 1. Regarding age, education, and WAIS scores, there were no significant main effects for diagnosis or treatment (all F<1), nor was there a significant interaction (age, F(1,42)=1.33, P=.255; education and WAIS, F<1). There were also no differences in gender makeup by treatment (P=.514) or diagnosis (P=1.000). The main effect of diagnosis was significant for the SPQ (F(1,42)=734.06, P<.001) and BAT (F(1,42)=828.67, P<.001). There was also no significant effect for treatment (SPQ, F(1,42)=2.16, P=.149; BAT score, F(1,42)=1.57, P=.218), nor was there a significant diagnosis x treatment interaction (SPQ, F(1,42)=2.54, P=.118; BAT score, F<1). The BDI-II was available for 42 participants (19 control [9 placebo,10 DCS]; 23 phobic [12 placebo, 11 DCS). All participants scored in the ‘normal’ range and there was no diagnosis (F(1,38)=2.84, P=.100) or treatment (F(1,38)=1.53, P=.224) main effect, and no significant diagnosis x treatment interaction (F<1).

The ASC was available for 35 participants (16 control [8 placebo, 8 DCS]; 19 phobic [10 placebo, 9 DCS]). For ASC score, neither the treatment main effect (F(1,31)=.63, P=.516), nor the diagnosis x treatment interaction (F(1,31)=1.65, P=.209) was significant. Compared to non-phobics, phobics reported more side effects (F(1,31)=7.93, P=.008) and were more likely to believe they received DCS (P=.017), but were not more accurate in their guess (P=.767). Thus, DCS was not associated with detectable side effects and participants were unable to detect whether they were given the active medication.

fMRI Behavioral Data

Repeated measures ANOVA revealed a significant main effect for diagnosis on SUDS for scan 1 and 2 (F(1,42)=671.70, P<.001) (Table 2). There were no significant differences in SUDS for treatment (F(1,42)=2.03, P=.162) or the diagnostic x treatment interaction (F(1,42)=1.83, P=.183). There was also no significant difference in SUDS between scan 1 and 2 (F(1,42)<1), indicating habituation did not occur.

There was a main effect for diagnosis on both spider- (F(1,42)=6.582, P=.014), and total-recognition discriminability (F(1,42)=6.02, P=.018), with phobics showing greater accuracy. There was no effect of diagnosis on butterfly-recognition (F(1,42)=.05, P=.825). Neither the treatment main effects nor the diagnosis x treatment interactions were significant (all F<1).

fMRI Analysis of Diagnostic Group

The diagnosis (phobic>control) x condition (spider>butterfly) interaction was examined within placebo groups (Table 3). There were significant differences between placebo-phobic and placebo-control groups (spider>butterfly) in the following a priori ROIs: right bed nucleus of the stria terminalis (BNST, extended amygdala), right insula, right parahippocampal gyrus, and left dlPFC. The condition main effect (spider>butterfly) was examined within each placebo group ( Table 4 and 5; Figures 1 and 2). The placebo phobic group showed activations in several ROIs, including left lateral amygdala, right dorsal and basomedial amygdala, right BNST, right insula, right dlPFC, and bilateral hippocampus. The placebo control group showed activations in a few ROIs, including the left lateral dorsal amygdala, right preamygdalar claustrum, right insula, and left OFC.

These results replicate findings from previous studies, with un-treated phobics showing unique activations in the bilateral dorsal and basomedial amygdala, right BNST, bilateral hippocampus, and right lateral PFC.46-48 Results support the use of this paradigm in examining DCS effects during phobic symptom provocation.

fMRI Analysis of DCS Effects

Results from the treatment (DCS>placebo) x condition (spider>butterfly) interaction are displayed in Tables 6 and 7 and Figures 1 and 2. DCS phobics had greater differential activations (spider>butterfly) than placebo phobics in the following a priori ROIs: left dlPFC, right dorsal ACC, bilateral inferior frontal gyrus (IFG), and bilateral insula. The corresponding group x condition interaction in controls revealed significant differences (spider>butterfly) in only one a priori ROI, the left ventral ACC.

Condition main effects (spider>butterfly) were examined within each DCS group (phobic, control). The DCS phobic group showed greater activations for spider versus butterfly in several a priori ROIs including: bilateral dlPFC, OFC, dorsal and ventral ACC, bilateral IFG, bilateral insula, and parahippocampal gyrus (PHG). Results are shown in Table 8 and Figure 1. In contrast to placebo phobics, DCS phobics did not have significant activation within the amygdala and had unique activations within OFC, ACC, and IFG.

The DCS control group showed greater activations for spider versus butterfly in the following a priori ROIs, all left lateralized: dlPFC, ventrolateral PFC (vlPFC), left lateral OFC, ACC, posterior cingulate, superior rostral gyrus, hippocampus, and PHG. Results are shown in Table 9 and Figure 2. In contrast to placebo controls, DCS controls did not show significant activation within the amygdala.

Behavioral Measures and fMRI Imaging Correlations

Only a priori regions significant for the spider > butterfly contrast within the phobic groups (DCS,placebo) were used for correlation analyses. For placebo-phobics, amygdala regions included the left lateral amygdala (x,y,z=-24,-4,-14), right dorsal amygdala (x,y,z=24,-4,-11), right basomedial amygdala (x,y,z=18,-1,-11), and right BNST (x,y,z=12,-1,-5). PFC regions included right vlPFC (x,y,z=45,32,10) and dlPFC (x,y,z=42,5,37). The right insula (x,y,z=36,2,1), and left (-x,y,z=24,-19,-14) and right hippocampus (x,y,z=27,-10,-14) were also used in these analyses. 

For DCS-phobics, the maximum voxel of the left BLA (x,y,z=-18,-1,-18) was used for correlation analyses. PFC regions included left (x,y,z=-24,44,28) and right dlPFC (x,y,z=45,35,13), left (x,y,z=-45, 20, 22) and right IFG (x,y,z=-45, 20, 22), and left (x,y,z=
-33,35,-11) and right OFC (x,y,z=45,17,-5). Additional ROIs included two regions of the dorsal ACC (Broadman area [BA] 24,x,y,z=6,2,46; BA 32,x,y,z=-3,17,31), left (x,y,z=-30,20,10) and right insula (x,y,z=36,2,4), and right PHG (x,y,z=24,-43,-8).

Within each group separately, ROIs were examined for correlations with fMRI SUDS, BAT, SPQ, and Spider D’. For placebo-phobics, there were significant correlations between SPQ and both right basomedial amygdala (r=.70, P=.011) and left vlPFC (r=.60, P=.038), and SUDS and left lateral amygdala (r=.65, P=.023).

In the DCS-phobics, there were significant correlations between SUDS and right OFC (r=.63, P=.039), BAT and right IFG (r=.72, P=.012), and Spider D’ and both right OFC (r=.74, P=.009) and left IFG (r=.64, P=.035).  

fMRI Imaging Correlations Between Prefrontal Cortex and Amygdala

In the placebo phobic group, there were positive correlations between right basomedial amygdala and both vlPFC (r=.76, P=.004) and dlPFC (r=.59, P=.045). For the DCS phobic group, there was a negative correlation between left BLA and left OFC (r=-.75, P=.008). However, one subject in the DCS phobic group was an outlier in this correlation and drove most of the effect. When this one subject was removed from the analysis, the correlation was no longer significant (r=-.31, P=.384).  

Discussion

The primary aim of this study was to investigate effects of DCS on brain activity during exposure to phobic stimuli in spider phobic and healthy control participants. Results are directly relevant to clinical findings that acute DCS enhances exposure therapy. We provide preliminary evidence that activity in regions within the PFC (dlPFC and IFG), dorsal ACC, and insula are enhanced by DCS during initial symptom provocation in specific phobia. The neural effects of DCS were less widespread among non-phobics, but did enhance activations in ventral ACC and caudate.
Findings of enhanced PFC activation are consistent with animal research showing DCS mediates medial PFC NMDA-receptor activity.15,50,51 However, the current study found DCS enhanced activation in primarily lateral PFC and OFC, as well as dorsal ACC. Lateral PFC and OFC have been associated with cognitive strategies to regulate emotion, such as reappraisal and distraction.52-54 The dorsal ACC is also involved in cognitive processing and is important in the allocation of attentional resources.55,56 DCS enhancement of these frontal regions may therefore reflect increased cognitive processing. DCS was also associated with increased insula activation in phobics. The insula has received increased focus in the anxiety disorders literature,57-59 and is consistently reported to activate during phobic symptom provocation48,49,60 and to normalize after treatment.34,61 It is understood to be involved in interoception and integration of internal body states with external events.56,57,62,63
Though not tested directly in this study, it is possible that increased PFC, OFC, ACC, and insula activation during exposure to phobic stimuli may serve as a mechanism for the ERP-enhancement effects of DCS found in clinical studies. In the animal literature, it has been suggested that DCS enhances generalization of extinction by enhancing the cognitive representation of the UCS, during exposure to the previously CS.7,68 Attention to the cognitive representation of the fear itself (rather than simply to the concrete stimuli) during exposure presumably relates to better treatment response.64-67 One interpretation of our findings is that increased activation in prefrontal and insular regions results in more complete integration of information regarding the fear experience and a stronger cognitive representation of the fear.  

In clinical research, it has been reported that DCS may influence the relationship between cognitive self-appraisals and exposure therapy outcome.18 Increased PFC activation could relate to such changes in cognitive appraisals as well as to the finding that DCS-augmented ERP may beneficially influence both anxiety and depressive symptoms.20 Future research should examine DCS effects on behavior and cognition during exposure therapy, and how these may relate to PFC, ACC, and insula activity.

Theories of DCS-enhanced fear extinction have focused primarily on the amygdala and hippocampus.5,6,8,13,68,70,71,72 In humans, DCS has been associated with decreased amygdala activation during facial processing23 and enhanced hippocampal activation during fear memory processing.24 In the current study, amygdala activation met criteria for significance only in placebo (not DCS) groups. However, the interaction analyses did not detect significant treatment effects on amygdala activation. It is possible that DCS does not modulate amygdala activation during symptom provocation; however, it is also possible the current study did not have adequate statistical power to detect DCS effects in this region.  

The current study identified correlations between brain regions of interest, and between regions of interest and clinical measures. The finding of a positive correlation between lateral PFC and amygdala activation in placebo-phobics is consistent with a previous report of positive correlations between vlPFC and amygdala in healthy controls during processing of surprised faces after presentation of negative sentences.73 This suggests that there may be reciprocal functional relationships between these regions during fear or anxiety processing. Clinical measures (eg, SUDS) positively correlated with amygdala activations in the placebo-phobic group. This finding replicates results from studies in other anxiety populations (eg, PTSD, anxiety-prone subjects)41,59 and is consistent with the role of the amygdala in fear and anxiety processing.25 Clinical measures and recognition accuracy for spider stimuli were correlated positively with prefrontal activations (eg, OFC) for DCS-phobics. As mentioned, lateral OFC has been proposed to mediate responses to negative affective states and excessive activation has been theorized to play a role in anxiety disorders.54 Thus, the finding of positive correlations between lateral OFC activation and clinical measures is not surprising, though the potential influence of DCS on this relationship should be further investigated.
Differences between the current study and the two previous acute-DCS neuroimaging studies may be due to differences in study population, medication dose, or paradigm. The two previous studies included only healthy, non-anxious participants,23,24 whereas we included an anxiety-disordered population. We administered DCS 100 mg whereas both previous studies used 500 mg. There is some indication that different doses of DCS have varying effects on neural and cognitive processes.22 Differences between studies may also be due to variations in fMRI paradigm and stage of fear processing examined. We examined effects of DCS at initial fear provocation, whereas Kalisch and colleagues examined more long-term effects during fear memory. DCS may initially modulate PFC activity (indicated in the current study), which translate into hippocampal modulation during later phases (indicated by Kalisch and colleagues).

Some limitations of this study should be considered. The sample size and statistical power were limited and therefore, current results should be considered preliminary until replicated by further research. Secondly, we did not include a CBT treatment arm, nor did we include extinction trials. As suggested, DCS may exert varying effects during different phases of the fear process. Future neuroimaging research should address this by using extinction paradigms involving prolonged exposure and by conducting scans pre- and post-DCS-augmented exposure therapy. Lastly, we did not include a perfusion measure (eg, arterial spin labeling) to investigate potential effects of DCS on the hemodynamic response. However, results from primate studies suggest DCS does not affect cardiovascular activity.74

 
Conclusion

The current study represents the first investigation of acute DCS effects on neural processing during symptom provocation in specific phobia. Results provide evidence that DCS enhances PFC, ACC, and insula activation during exposure to phobic stimuli. These effects may relate to findings from clinical studies showing acute-DCS to enhance exposure therapy for anxiety disorders.16-18,20 Future animal and human research should further investigate how DCS modulates activity in the PFC. The clinical significance of these effects should be investigated as they relate to changes in emotion, cognition, and behavior during exposure therapy. CNS 

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